A molecular and structural approach to understanding interstrand crosslink incision by the Fanconi anaemia DNA repair pathway

Our genetic blueprint is contained within long, chromosomal molecules of DNA in the nucleus of our cells. During our lifetime, the cells in many of our tissues are constantly dividing to replace old and damaged cells. This is part of the natural ageing process. Before dividing, a cell must replicate its DNA accurately to prevent chromosomal changes that could lead to debilitating degenerative diseases including cancer and neurodegeneration, many of which are hallmarks of ageing. DNA replication is performed by DNA polymerases that copy the template strands in the parent cell, as part of full chromosome duplication and cell division. Since the DNA is composed of two strands of DNA, replication involves separation and replication of these two strands, and replication of each strand in a co-ordinated process requiring dedicated factors for each strand. When the DNA strands are separated the structure produced contains unwound regions, and the junction where unwinding and replication are ongoing is named a 'replication fork'. Importantly, during every round of replication the dedicated replication proteins encounter structures or damage within the DNA that block their progress. These structures might be naturally arising regions of the DNA that are 'hard' to replicate, or they might be chemically damaged regions within DNA. Such chemical damage can arise spontaneously as a result of normal cellular processes that are constantly occurring, or they might be inflicted through exposure to external agents, for example solar radiation (sunlight) or a variety of environmental chemical agents. Moreover, this type of damage is produced is also produced by several important medicines used to treat cancer, and a full understanding of how cells respond to this damage might help us improve chemotherapy.

The abnormalities encountered during replication must be repaired, and this frequently involves proteins called endonucleases, that cut abnormal DNA structures. This can occur either during the process of replication, or structures generated during DNA replication can be later resolved after replication is complete. Endonucleases initiate this cascade of repair events, and several key factors known to be required for are the XPF-ERCC1, SLX1 and MUS81-EME1 proteins. However, it has recently become apparent that these factors must be associated with a large 'platform' protein called SLX4 which helps direct it to the damaged replication forks, and other structures associated with the repair of damaged DNA. Solving the three-dimensional structure of the SLX4 complex at high resolution will be key to understanding its mechanism. This in turn will ultimately help in the development of new therapeutics combating a number of degenerative conditions associated with ageing. It should also improve our diagnosis of developmental and malignant disorders and provide important insights that the pharmaceutical and biotechnology sectors could use to generate new medicines and technologies, especially since it appears that targeted inhibition of these repair reactions might help improve cancer therapy.

Loss of genomic stability elicits disease and is a trigger for human ageing. During every cell division, structural aberrations in the DNA can block the progression of DNA replication. The inherited syndrome Fanconi anaemia (FA) produces devastating and often lethal clinical features including bone marrow failure, developmental defects and solid and haematological malignancies. The cellular defect in FA is an inability to purge the genome of DNA interstrand crosslinks (ICLs), using the 'FA' DNA repair pathway. ICLs are amongst the most toxic forms of DNA damage known and arise when duplex DNA strands become covalently linked.

Despite advances in identifying key components and landmark steps, key molecular details of how the FA pathway co-ordinate to ICL processing removal are obscure. Here, we will bring together a powerful combination of nucleic acid chemistry, protein biochemistry with structural and molecular biology to reveal the mechanistic details of FA pathway operation. We will focus on the pivotal step of ICL incision by the DNA cutting enzyme XPF-ERCC1 and how the localisation of this complex is regulated by additional key FA proteins including FAND2 and FANCI. Our approach will allow us to iteratively test the functional importance of the structural features identified using biochemical DNA repair reactions.

By studying this repair pathway in unprecedented detail and depth, we will transform our understanding of the FA and open-up new avenues to treat affected patients. The genes defective in FA are also associated with predisposition to disease in the population more generally and represent attractive therapeutic targets, including in cancer treatment. Our work will, therefore, be vital to enable the targeting FA DNA repair factors in a range of human diseases.